- Email: [email protected]
Microbial metabolism of 2-deoxyglucose
204
PRELIMINARY NOTES
phate or glycerol in the presence and absence of high concentrations of exogenous glucose. No differences were observed (unpublished data). Regardless of the metabolic basis for the effect, exogenous glucose at concentrations as low as IO ~t M can exert a differential effect on the various pathways leading to zaC02 from C-I and C-6. In this system as in others, this effect m a y be further complicated by permeability differences in various tissues or at various stages of development. This work was supported b y Grant GM-o8958 from the National Institutes of Health, U.S. Public Health Service. This is publication No. oooo of the Cancer Commission of H a r v a r d University.
J. Collins Warren Laboratories, Harvard University, Massachusetts General Hospital, Boston, Mass. (U.S.A.)
B. E. WRIGHT M. BRUHMULLER
t j . KATZ AND 1-I. G. WOOD, J. Biol. Chem., 235 (I96O) 2165. 2 V. t-I. CHELDI~LIN, Metabolic Pathways in Microorganisms, J o h n W i l e y & Sons, N e w York, I 9 6 I . a M. E, KRAHL, Biochim. Biophys. Acta, 20 (1956) 27. 4 L. FRIDHANDLER, Exptl. Cell Res., 22 (1961) 303 . 5 g . E. WRIGHT, M. BRUHMULLER AND C. WARD, Develop. Biol., s u b m i t t e d for p u b l i c a t i o n . 8 B. E. WRIGHT AND M. I~RUHMULLIgR, Federation Proc., (1963) 584 . T B. E. WRIGHT AND B. BLOOM, Biochim. Biophys. ,4cta, 46 (1961) 342.
Received November Ist, 1963 Biochim. Biophys. ,.~cta, 82 (I964) 203-204
PN 1333
Microbial metabolism of 2-deoxyglucose 2-Deoxy-D-glucose is known to inhibit the metabolism and growth of m a n y organismsl, 2. Studies on the metabolism of 2-deoxyglucose have shown that it can be phosphorylated to 2-deoxyglucose 6-phosphate in the presence of hexokinase (EC 2.7.1.1 ) and ATP 2, or it can be oxidized to 2-deoxygluconic acidS, ~. Except for these limited observations, there have apparently been no reports on the intermediary metabolism of 2-deoxyglucose; the present study was undertaken to elucidate the pathway of metabolism of 2-deoxyglucose in an organism which could utilize this sugar as a carbon and energy source. The unidentified pseudomonad used in these studies was isolated from soil by selective enrichment techniques. During growth on 2-deoxyglucose, the concentration of reducing sugar decreased 5, and an aldonic acid was detected in the culture fluid 6. This compound was also produced by cell-free extracts, which were prepared by disruption of cell suspensions in a French pressure cell. To aid. in the identification of the product, 2-deoxygluconolactone and the acid were prepared by bromine oxidation of 2-deoxyglucose. Although it had been reported that the bromine oxidation of 2-deoxyglucose 6-phosphate yielded 6-phosphomannonic acid 7, the product mentioned above was clearly distinguished by paper chromatography in several solvent systems from D-mannonolactone (obtained from Dr. G. ASHWELL). For the purpose Biochim. Biophys. Acta, 82 (1964) 204 207
PRELIMINARY NOTES
205
of identifying the metabolic product, tritium-labeled 2-deoxyglucose was prepared by incubation of the sugar in 3I-I~0 at pFI 8 (performed by New England Nuclear Corporation). Presumably the tritium is incorporated by an alkali-catalyzed exchange at C-2. Fig. i demonstrates the conversion of tritiated 2-deoxyglucose to 2-deoxygluconic acid and 2-deoxygluconolactone by a cell-free extract. An aliquot of the (A~ 0 time control
×
,
._c E 4O
g 18o
o
B) Incubated 4 h
140 ._> 100 u
6O e~
20 O
T 2-Deoxygluconic acid
o' 4' ; ' ; 2
2_DTxy-T2_ Deoxyglucono , glucose lactone
~6'2o'2'4'~8'~2'
centimeters
Fig. I. C h r o m a t o g r a p h y of e n z y m a t i c o x i d a t i o n p r o d u c t of t r i t i a t e d 2-deoxyglucose. R e a c t i o n m i x t u r e contained, in a t o t a l v o l u m e of 2 m l : 0 . 9 / , m o l e 2-deoxy[3H]glucose (specific a c t i v i t y , 8.8-1o s counts/min/ffmole), io ffmoles MgCI~, 27. 5/*moles p o t a s s i u m p h o s p h a t e buffer (pH 7.1) a n d 0.6 ml of extract.
deproteinized reaction mixture was subjected to descending paper chromatography (pyridine-n-butanol-H20; 4: 6:3, v/v). The chromatographed strip was dried and cut into pieces I.o cm in length. These were analyzed for radioactivity in a NuclearChicago Model-?25 liquid-scintillation counter by placing each i.o-cm section in a counting vial containing scintillation fluid*. Approx. one-third of the 2-deoxyglucose added to the incubated tube was converted to 2-deoxygluconic acid. The ease with which 2-deoxygluconic acid and its lactone are interconverted non-enzymatically prevents a choice between the two as the oxidation product, although it is probably the lactone which is produced by the oxidation of the pyranose ring. This reaction is 02-dependent, but does not require added cofactors. Upon incubation of resting cells with 2-deoxygluconic acid, approx, one-third of the substrate disappeared after 4 h. When crude extracts were incubated with 2-deoxygluconic acid, two results were observed: first, a decrease in substrate concentration, and secondly, the appearance of a reducing compound as determined by the Nelson test. This reaction was markedly stimulated by the addition of NAD and * T h e scintillation fluid consisted of 4 ° g of n a p h t h a l e n e , 14 g of 2,5-diphenyloxazole, a n d 6o m g of p-bis[2-(5-phenyloxazolyl)]benzene in 16o ml of dioxane.
Biochim. Biophys. Acta, 82 (1964) 2o4-2o 7
206
PRELIMINARY NOTES
FMN. The enzymatic activity for this reaction is in the IOOOOO × g supematant solution and is not affected b y dialysis. In order to identify the reducing compound formed in this reaction, a large-scale incubation was carried out in which 200/~moles of potassium 2-deoxygluconate, 50/,moles of NAD and I mmole of potassium phosphate buffer (pH 7,1) were incubated with IO ml of extract for 4 h. When the deproteinized incubation mixtures were applied to Dowex-I (acetate form) columns, the effluent from the incubated sample was found to contain a neutral carbonyl compound which was absent from the zero-time control. On the basis of its chromatographic properties in two solvent systems (pyridine-n-butanol-water; 4: 6: 3, v/v; n-butanol-acetic acid-water; 4: I : 5, v/v) and of the melting point of its 2, 4-dinitrophenylhydrazone derivative (I53°-I56°), the compound was identified as glyceraldehyde. In consideration of the structure of the substrate, it is possible that the glyceraldehyde arises from the bottom three carbon atoms of 2-deoxygluconate, in which case one would expect the glyceraldehyde to be of the D- configuration. TABLE [ FORMATION
OF
"KF.TO ACID"
Complete s y s t e m contains io umoles p o t a s s i u m 2-deoxygluconate, 2.5/~inoles NAD, i /zmole FMN. 5 ° #moles p o t a s s i u m p h o s p h a t e buffer (pH 7.1) and 0.5 ml of e x t r a c t in 2 ml total volume. Carbonyl compound (pmoles/ml) 3o-min incubation
Complete Zero time minus FMN minus potassium 2-deoxygluconate
4-h incubation
Total
Dowex effuent
Total
Dowex effuent
6.7 4.2 5.3
i. i 0.3 0.7
8.7 4.2 3-3
3-5 0.3 o.9
4.2
0.3
4.2
o.3
Since it might be expected that there would be one or more intermediates between 2-deoxygluconate and glyceraldehyde, attempts were made to detect such intermediates after shorter incubation periods, Table I shows that incubation of the crude extract for 30 min with NAD and 2-deoxygluconate results in the formation of an anionic reducing compound as indicated by a positive reaction in the Nelson test and retention b y Dowex-I (acetate form). The amount present at zero time is due to NAD which yields a positive reaction in the Nelson test. Longer incubations in the presence of both NAD and FMN led to accumulation of a neutral carbonyl compound, presumably glyceraldehyde, as shown above. I t is possible that the acidic carbonyl compound is a keto-2-deoxygluconic acid, and that the glyceraldehyde is a cleavage product of the metabolism of this intermediate. Present investigations are concerned with the isolation and identification of the presumed intermediate and with the enzymatic reactions involved in its formation and subsequent metabolism. This research was supported in part by Grant No. 423 from the Massachusetts Heart Association and by Grant No. AI-o 3 586 from the National Institutes of Health, U.S. Public Health Service. A brief report of this work has been presented 12. M.M.E. Biochim. Biophys, Acta, 82 (1964) 2o 4 2o 7
207
PRELIMINARY NOTES
is a Predoctoral Research Fellow, Division of General Medical Sciences, U.S. Public Health Service. M.A.C. is a Research Career Development Awardee, U.S. Public Health Service.
Department of Biochemistry, Tufts University School of Medicine, Boston, Mass. (U.S.A.)
MARY M. EICHHORN
MORRIS A. CYNKIN
V. B. CRAMER AND G. E. WOODWARD, J. Franklin Inst., 253 (1952) 354A. N. WICK, D. R. DRURY, H. I. NAKADA AND J. B. WOLFE, J. Biol. Chem., 224 (1957) 963. 3 A. K. WILLIAMS AND R. G. EAGON, J. Bacteriol., 77 (1959) 167. 4 E. R. BLAKLEY AND O. CIFERRI, Can. J. Microbiol., 5 (1961) 61. 5 N. NELSON, J. Biol. Chem., 153 (1944) 375. 8 S. HESTRIN, J. Biol. Chem., 18o (1949) 249. 7 S. A. BROOKS, J. C. LAWRENCE AND C. R. RICKETTS, Nature, 187 (196o) lO28. s M. M. EICHHORN AND M. A. CYNKIN,Am. Chem. Soc. Abstr. (5-C), September 1963. 1
Received October 25th, 1963 Biochim. Biophys. Acta, 82 (1964) 2o4-2o 7
PN 1332
Catabolism of ~-hydroxy[2-14C]glutamic acid by excised leaves of Phlox decussata The mammalian metabolism of y-hydroxygiutamate, an intermediate in hydroxyproline catabolism l, 2, has recently been described in considerable detail 3-~°. Studies in our laboratory have shown that enzymes present in rat-liver extracts catalyze the net formation of glyoxylate and alanine from y-hydroxyglutamate by the following sequence of reaction#, n : y - h y d r o x y g l u t a m a t e + a-ketoglutarate --~ a - k e t o - y - h y d r o x y g l u t a r a t e + g l u t a m a t e ~-keto-y-hydroxyglutarate ~ p y r u v a t e + glyoxylate p y r u v a t e + g l u t a m a t e - ~ a-ketoglutarate + alanine N e t reaction: ~,-hydroxyglutamate ~ glyoxylate + alanine
No information is available, however, concerning the synthesis and breakdown of this amino acid by Phlox decussata, the plant in which it was originally discovered 12 and where it occurs as a maior constituent of the soluble nitrogen fraction of the leaf 1~. In experiments designed to study the formation of ~-hydroxygiutamate in Phlox, other investigators 14,15 detected no assimilation of isotope into ~-hydroxyglutamate after administering ~4C02 and E2A4C3pyruvate to the plant. The suggestion has been made, therefore, that the compound might represent only a storage product. These findings led us to examine the possibility of whether or not the plant is able to degrade ~-hydroxyglutamate. The ~,-hydroxy~2-14Cjglutamate used in these studies was synthesized by the method of BENOITON AXI) BOUTI-IILLIER3 (specific activity-----103000 counts/rain per /,mole). The compound migrated on one- and two-dimensional chromatograms Biochim. Biophys. Acta, 82 (1964) 2o7-2o 9
PRELIMINARY NOTES
phate or glycerol in the presence and absence of high concentrations of exogenous glucose. No differences were observed (unpublished data). Regardless of the metabolic basis for the effect, exogenous glucose at concentrations as low as IO ~t M can exert a differential effect on the various pathways leading to zaC02 from C-I and C-6. In this system as in others, this effect m a y be further complicated by permeability differences in various tissues or at various stages of development. This work was supported b y Grant GM-o8958 from the National Institutes of Health, U.S. Public Health Service. This is publication No. oooo of the Cancer Commission of H a r v a r d University.
J. Collins Warren Laboratories, Harvard University, Massachusetts General Hospital, Boston, Mass. (U.S.A.)
B. E. WRIGHT M. BRUHMULLER
t j . KATZ AND 1-I. G. WOOD, J. Biol. Chem., 235 (I96O) 2165. 2 V. t-I. CHELDI~LIN, Metabolic Pathways in Microorganisms, J o h n W i l e y & Sons, N e w York, I 9 6 I . a M. E, KRAHL, Biochim. Biophys. Acta, 20 (1956) 27. 4 L. FRIDHANDLER, Exptl. Cell Res., 22 (1961) 303 . 5 g . E. WRIGHT, M. BRUHMULLER AND C. WARD, Develop. Biol., s u b m i t t e d for p u b l i c a t i o n . 8 B. E. WRIGHT AND M. I~RUHMULLIgR, Federation Proc., (1963) 584 . T B. E. WRIGHT AND B. BLOOM, Biochim. Biophys. ,4cta, 46 (1961) 342.
Received November Ist, 1963 Biochim. Biophys. ,.~cta, 82 (I964) 203-204
PN 1333
Microbial metabolism of 2-deoxyglucose 2-Deoxy-D-glucose is known to inhibit the metabolism and growth of m a n y organismsl, 2. Studies on the metabolism of 2-deoxyglucose have shown that it can be phosphorylated to 2-deoxyglucose 6-phosphate in the presence of hexokinase (EC 2.7.1.1 ) and ATP 2, or it can be oxidized to 2-deoxygluconic acidS, ~. Except for these limited observations, there have apparently been no reports on the intermediary metabolism of 2-deoxyglucose; the present study was undertaken to elucidate the pathway of metabolism of 2-deoxyglucose in an organism which could utilize this sugar as a carbon and energy source. The unidentified pseudomonad used in these studies was isolated from soil by selective enrichment techniques. During growth on 2-deoxyglucose, the concentration of reducing sugar decreased 5, and an aldonic acid was detected in the culture fluid 6. This compound was also produced by cell-free extracts, which were prepared by disruption of cell suspensions in a French pressure cell. To aid. in the identification of the product, 2-deoxygluconolactone and the acid were prepared by bromine oxidation of 2-deoxyglucose. Although it had been reported that the bromine oxidation of 2-deoxyglucose 6-phosphate yielded 6-phosphomannonic acid 7, the product mentioned above was clearly distinguished by paper chromatography in several solvent systems from D-mannonolactone (obtained from Dr. G. ASHWELL). For the purpose Biochim. Biophys. Acta, 82 (1964) 204 207
PRELIMINARY NOTES
205
of identifying the metabolic product, tritium-labeled 2-deoxyglucose was prepared by incubation of the sugar in 3I-I~0 at pFI 8 (performed by New England Nuclear Corporation). Presumably the tritium is incorporated by an alkali-catalyzed exchange at C-2. Fig. i demonstrates the conversion of tritiated 2-deoxyglucose to 2-deoxygluconic acid and 2-deoxygluconolactone by a cell-free extract. An aliquot of the (A~ 0 time control
×
,
._c E 4O
g 18o
o
B) Incubated 4 h
140 ._> 100 u
6O e~
20 O
T 2-Deoxygluconic acid
o' 4' ; ' ; 2
2_DTxy-T2_ Deoxyglucono , glucose lactone
~6'2o'2'4'~8'~2'
centimeters
Fig. I. C h r o m a t o g r a p h y of e n z y m a t i c o x i d a t i o n p r o d u c t of t r i t i a t e d 2-deoxyglucose. R e a c t i o n m i x t u r e contained, in a t o t a l v o l u m e of 2 m l : 0 . 9 / , m o l e 2-deoxy[3H]glucose (specific a c t i v i t y , 8.8-1o s counts/min/ffmole), io ffmoles MgCI~, 27. 5/*moles p o t a s s i u m p h o s p h a t e buffer (pH 7.1) a n d 0.6 ml of extract.
deproteinized reaction mixture was subjected to descending paper chromatography (pyridine-n-butanol-H20; 4: 6:3, v/v). The chromatographed strip was dried and cut into pieces I.o cm in length. These were analyzed for radioactivity in a NuclearChicago Model-?25 liquid-scintillation counter by placing each i.o-cm section in a counting vial containing scintillation fluid*. Approx. one-third of the 2-deoxyglucose added to the incubated tube was converted to 2-deoxygluconic acid. The ease with which 2-deoxygluconic acid and its lactone are interconverted non-enzymatically prevents a choice between the two as the oxidation product, although it is probably the lactone which is produced by the oxidation of the pyranose ring. This reaction is 02-dependent, but does not require added cofactors. Upon incubation of resting cells with 2-deoxygluconic acid, approx, one-third of the substrate disappeared after 4 h. When crude extracts were incubated with 2-deoxygluconic acid, two results were observed: first, a decrease in substrate concentration, and secondly, the appearance of a reducing compound as determined by the Nelson test. This reaction was markedly stimulated by the addition of NAD and * T h e scintillation fluid consisted of 4 ° g of n a p h t h a l e n e , 14 g of 2,5-diphenyloxazole, a n d 6o m g of p-bis[2-(5-phenyloxazolyl)]benzene in 16o ml of dioxane.
Biochim. Biophys. Acta, 82 (1964) 2o4-2o 7
206
PRELIMINARY NOTES
FMN. The enzymatic activity for this reaction is in the IOOOOO × g supematant solution and is not affected b y dialysis. In order to identify the reducing compound formed in this reaction, a large-scale incubation was carried out in which 200/~moles of potassium 2-deoxygluconate, 50/,moles of NAD and I mmole of potassium phosphate buffer (pH 7,1) were incubated with IO ml of extract for 4 h. When the deproteinized incubation mixtures were applied to Dowex-I (acetate form) columns, the effluent from the incubated sample was found to contain a neutral carbonyl compound which was absent from the zero-time control. On the basis of its chromatographic properties in two solvent systems (pyridine-n-butanol-water; 4: 6: 3, v/v; n-butanol-acetic acid-water; 4: I : 5, v/v) and of the melting point of its 2, 4-dinitrophenylhydrazone derivative (I53°-I56°), the compound was identified as glyceraldehyde. In consideration of the structure of the substrate, it is possible that the glyceraldehyde arises from the bottom three carbon atoms of 2-deoxygluconate, in which case one would expect the glyceraldehyde to be of the D- configuration. TABLE [ FORMATION
OF
"KF.TO ACID"
Complete s y s t e m contains io umoles p o t a s s i u m 2-deoxygluconate, 2.5/~inoles NAD, i /zmole FMN. 5 ° #moles p o t a s s i u m p h o s p h a t e buffer (pH 7.1) and 0.5 ml of e x t r a c t in 2 ml total volume. Carbonyl compound (pmoles/ml) 3o-min incubation
Complete Zero time minus FMN minus potassium 2-deoxygluconate
4-h incubation
Total
Dowex effuent
Total
Dowex effuent
6.7 4.2 5.3
i. i 0.3 0.7
8.7 4.2 3-3
3-5 0.3 o.9
4.2
0.3
4.2
o.3
Since it might be expected that there would be one or more intermediates between 2-deoxygluconate and glyceraldehyde, attempts were made to detect such intermediates after shorter incubation periods, Table I shows that incubation of the crude extract for 30 min with NAD and 2-deoxygluconate results in the formation of an anionic reducing compound as indicated by a positive reaction in the Nelson test and retention b y Dowex-I (acetate form). The amount present at zero time is due to NAD which yields a positive reaction in the Nelson test. Longer incubations in the presence of both NAD and FMN led to accumulation of a neutral carbonyl compound, presumably glyceraldehyde, as shown above. I t is possible that the acidic carbonyl compound is a keto-2-deoxygluconic acid, and that the glyceraldehyde is a cleavage product of the metabolism of this intermediate. Present investigations are concerned with the isolation and identification of the presumed intermediate and with the enzymatic reactions involved in its formation and subsequent metabolism. This research was supported in part by Grant No. 423 from the Massachusetts Heart Association and by Grant No. AI-o 3 586 from the National Institutes of Health, U.S. Public Health Service. A brief report of this work has been presented 12. M.M.E. Biochim. Biophys, Acta, 82 (1964) 2o 4 2o 7
207
PRELIMINARY NOTES
is a Predoctoral Research Fellow, Division of General Medical Sciences, U.S. Public Health Service. M.A.C. is a Research Career Development Awardee, U.S. Public Health Service.
Department of Biochemistry, Tufts University School of Medicine, Boston, Mass. (U.S.A.)
MARY M. EICHHORN
MORRIS A. CYNKIN
V. B. CRAMER AND G. E. WOODWARD, J. Franklin Inst., 253 (1952) 354A. N. WICK, D. R. DRURY, H. I. NAKADA AND J. B. WOLFE, J. Biol. Chem., 224 (1957) 963. 3 A. K. WILLIAMS AND R. G. EAGON, J. Bacteriol., 77 (1959) 167. 4 E. R. BLAKLEY AND O. CIFERRI, Can. J. Microbiol., 5 (1961) 61. 5 N. NELSON, J. Biol. Chem., 153 (1944) 375. 8 S. HESTRIN, J. Biol. Chem., 18o (1949) 249. 7 S. A. BROOKS, J. C. LAWRENCE AND C. R. RICKETTS, Nature, 187 (196o) lO28. s M. M. EICHHORN AND M. A. CYNKIN,Am. Chem. Soc. Abstr. (5-C), September 1963. 1
Received October 25th, 1963 Biochim. Biophys. Acta, 82 (1964) 2o4-2o 7
PN 1332
Catabolism of ~-hydroxy[2-14C]glutamic acid by excised leaves of Phlox decussata The mammalian metabolism of y-hydroxygiutamate, an intermediate in hydroxyproline catabolism l, 2, has recently been described in considerable detail 3-~°. Studies in our laboratory have shown that enzymes present in rat-liver extracts catalyze the net formation of glyoxylate and alanine from y-hydroxyglutamate by the following sequence of reaction#, n : y - h y d r o x y g l u t a m a t e + a-ketoglutarate --~ a - k e t o - y - h y d r o x y g l u t a r a t e + g l u t a m a t e ~-keto-y-hydroxyglutarate ~ p y r u v a t e + glyoxylate p y r u v a t e + g l u t a m a t e - ~ a-ketoglutarate + alanine N e t reaction: ~,-hydroxyglutamate ~ glyoxylate + alanine
No information is available, however, concerning the synthesis and breakdown of this amino acid by Phlox decussata, the plant in which it was originally discovered 12 and where it occurs as a maior constituent of the soluble nitrogen fraction of the leaf 1~. In experiments designed to study the formation of ~-hydroxygiutamate in Phlox, other investigators 14,15 detected no assimilation of isotope into ~-hydroxyglutamate after administering ~4C02 and E2A4C3pyruvate to the plant. The suggestion has been made, therefore, that the compound might represent only a storage product. These findings led us to examine the possibility of whether or not the plant is able to degrade ~-hydroxyglutamate. The ~,-hydroxy~2-14Cjglutamate used in these studies was synthesized by the method of BENOITON AXI) BOUTI-IILLIER3 (specific activity-----103000 counts/rain per /,mole). The compound migrated on one- and two-dimensional chromatograms Biochim. Biophys. Acta, 82 (1964) 2o7-2o 9